Inter-simple sequence repeat (ISSR) analysis of genetic variation of Chondrus crispus populations from North Atlantic

ISSR analysis was used to investigate genetic variations of 184 haploid and diploid samples from nine North Atlantic Chondrus crispus Stackhouse populations and one outgroup Yellow Sea Chondrus ocellatus Holmes population. Twenty-two of 50 primers were selected and 163 loci were scored for genetic diversity analysis. Genetic diversity varied among populations, percentage of polymorphic bands (PPB) ranged from 27.0 to 55.8%, H(Nei's genetic diversity) ranged from 0.11 to 0.20 and I(Shannon's information index) ranged from 0.16 to 0.30. Estimators PPB, H and I had similar values in intra-population genetic diversity, regardless of calculation methods. Analysis of molecular variance (AMOVA) apportioned inter-population and intra-population variations for C crispus, showing more genetic variance (56.5%) occurred in intra-population, and 43.5% variation among nine populations. The Mantel test suggested that genetic differentiation between nine C. crispus populations was closely related with geographic distances (R = 0.78, P = 0.002). Results suggest that, on larger distance scale (ca. > 1000 km), ISSR analysis is useful for determining genetic differentiations of C crispus populations including morphologically inseparable haploid and diploid individuals. (c) 2007 Elsevier B.V. All rights reserved.

Species identification in salted products of red snappers by semi-nested PCR-RFLP based on the mitochondria 12S rRNA gene sequence

A molecular approach was developed to distinguish species of red snappers among commercial salted fish products. The specific fragments of the mitocliondrial 12S rRNA gene, which were about 450 bp, were obtained using the semi-nested polymerase chain reaction (semi-nested PCR). Subsequently, PCR amplicons were sequenced, aiming to select restriction endonucleases that generated species-specific restriction fragment length polymorphism (RFLP) profiles. Discrimination of red snappers Lutjanus sanguineus, L. erythopterus from L. argentintaculatus, L. malabarius and other morphologically similar fishes such as Lethrinus leutjanus and Pinjalo pinjalo was feasible by one restriction digestion reaction with three endonucleases Hae III, Sca I and SnaB I, however, for differentiation of L. sattguineus and L. erythopterus, another restriction digestion reaction with single restriction endonuclease Mae II was needed. The seminested PCR-RFLP was demonstrated to be reliable in species identification of salted fish products in this study. (c) 2005 Published by Elsevier Ltd.

Sequence analysis and functional study of thymidylate synthase from zebrafish, Danio rerio

The thymidylate synthase (TS), an important target for many anticancer drugs, has been cloned from different species. But the cDNA property and function of TS in zebrafish are not well documented. In order to use zebrafish as an animal model for screening novel anticancer agents, we isolated TS cDNA from zebrafish and compared its sequence with those from other species. The open reading frame (ORF) of zebrafish TS cDNA sequence was 954 nucleotides, encoding a 318-amino acid protein with a calculated molecular mass of 36.15 kDa. The deduced amino acid sequence of zebrafish TS was similar to those from other organisms, including rat, mouse and humans. The zebrafish TS protein was expressed in Escherichia coli and purified to homogeneity. The purified zebrafish TS showed maximal activity at 28 degrees C with similar K-m value to human TS. Western immunoblot assay confirmed that TS was expressed in all the developmental stages of zebrafish with a high level of expression at the 1-4 cell stages. To study the function of TS in zebrafish embryo development, a short hairpin RNA (shRNA) expression vector, pSilencer 4.1-CMV/TS, was constructed which targeted the protein-coding region of zebrafish TS mRNA. Significant change in the development of tail and epiboly was found in zebrafish embryos microinjected pSilencer4.1-CMV/TS siRNA expression vector.

PenBase, the shrimp antimicrobial peptide penaeidin database: Sequence-based classification and recommended nomenclature

Antimicrobial peptides play a major role in innate immunity. The penaeidins, initially characterized from the shrimp Litopenaeus vannamei, are a family of antimicrobial peptides that appear to be expressed in all penaeid shrimps. As of recent, a large number of penaeid nucleotide sequences have been identified from a variety of penaeid shrimp species and these sequences currently reside in several databases under unique identifiers with no nomenclatural continuity. To facilitate research in this field and avoid potential confusion due to a diverse number of nomenclatural designations, we have made a systematic effort to collect, analyse, and classify all the penaeidin sequences available in every database. We have identified a common penaeidin signature and subsequently established a classification based on amino acid sequences. In order to clarify the naming process, we have introduced a 'penaeidin nomenclature' that can be applied to all extant and future penaeidins. A specialized database, PenBase, which is freely available at http://www.penbase.immunaqua.com, has been developed for the penaeidin family of antimicrobial peptides, to provide comprehensive information about their properties, diversity and nomenclature. (c) 2005 Elsevier Ltd. All rights reserved.

Development of expressed sequence tags from the bay scallop, Argopecten irradians irradians

The bay scallop, Argopecten irradians irradians, introduced from North America, has become one of the most important aquaculture species in China. Inan effort to identify scallop genes involved in host defense, a high-quality cDNA library was constructed from whole body tissues of the bay scallop. A total of 5828 successful sequencing reactions yielded 4995 expressed sequence tags (ESTs) longer than 100 bp. Cluster and assembly analyses of the ESTs identified 637 contigs (consisting of 2853 sequences) and 2142 singletons, totaling 2779 unique sequences. Basic Local Alignment Search Tool (BLAST) analysis showed that the majority (73%) of the unique sequences had no significant homology (E-value >= 0.005) to sequences in GenBank. Among the 748 sequences with significant GenBank matches, 160 (21.4%) were for genes related to metabolism, 131 (17.5%) for cell/organism defense, 124 (16.6%) for gene/protein expression, 83 (11.1%) for cell structure/motility, 70 (9.4%) for cell signaling/communication, 17 (2.3%) for cell division, and 163 (21.8%) matched to genes of unknown functions. The list of host-defense genes included many genes with known and important roles in innate defense such as lectins, defensins, proteases, protease inhibitors, heat shock proteins, antioxidants, and Toll-like receptors. The study provides a significant number of ESTs for gene discovery and candidate genes for studying host defense in scallops and other molluscs.

Ulva and Enteromorpha (Ulvaceae, Chlorophyta) from two sides of the Yellow Sea: analysis of nuclear rDNA ITS and plastid rbcL sequence data

Ulvacean green seaweeds are common worldwide; they formed massive green tides in the Yellow Sea in recent years, which caused marine ecological problems as well as a social issue. We investigated two major genera of the Ulvaceae, Ulva and Enteromorpha, and collected the plastid rbcL and nuclear ITS sequences of specimens of the genera in two sides of the Yellow Sea and analyzed them. Phylogenetic trees of rbcL data show the occurrence of five species of Enteromorpha (E. compressa, E. flexuosa, E. intestinalis, E. linza and E. prolifera) and three species of Ulva (U. pertusa, U. rigida and U. ohnoi). However, we found U. ohnoi, which is known as a subtropical to tropical species, at two sites on Jeju Island, Korea. Four ribotypes in partial sequences of 5.8S rDNA and ITS2 from E. compressa were also found. Ribotype network analysis revealed that the common ribotype, occurring in China, Korea and Europe, is connected with ribotypes from Europe and China/Japan. Although samples of the same species were collected from both sides of the Yellow Sea, intraspecific genetic polymorphism of each species was low among samples collected worldwide.

Species identification in salted products of red snappers by semi-nested PCR-RFLP based on the mitochondrial 12S rRNA gene sequence

A molecular approach was developed to distinguish species of red snappers among commercial salted fish products. The specific fragments of the mitochondrial 12S rRNA gene, which were about 450bp, were obtained using the semi-nested polymerase chain reaction (semi-nested PCR). Subsequently, PCR arnplicons were sequenced, aiming to select restriction endonucleases that generated species-specific restriction fragment length polymorphism (RFLP) profiles. Discrimination of red snappers Lutjanus sanguineus, Lutjanus erythopterus from Lutjanus argentimaculatus, Lutjanus malabarius and other morphologically similar fishes such as Lethrinus leutjanus and Pinjalo pinjalo was feasible by one restriction digestion reaction with three endonucleases Hae III, Sca I and SnaB I, however, for discrimination of L. sanguineus and L. erythopterus, another restriction digestion reaction with single restriction endonuclease Mae II was needed. The semi-nested PCR-RFLP was demonstrated to be reliable in species identification of salted fish products in this study. (c) 2005 Elsevier Ltd. All rights reserved.

Characterization and mapping of expressed sequence tags isolated from a subtracted cDNA library of Litopenaeus vannamei injected with white spot syndrome virus

A large number of polymorphic simple sequence repeats (SSRs) or microsatellites are needed to develop a genetic map for shrimp. However, developing an SSR map is very time-consuming, expensive, and most SSRs are not specifically linked to gene loci of immediate interest. We report here on our strategy to develop polymorphic markers using expressed sequence tags (ESTs) by designing primers flanking single or multiple SSRs with three or more repeats. A subtracted cDNA library was prepared using RNA from specific pathogen-free (SPF) Litopenaeus vannamei juveniles (similar to 1 g) collected before (0) and after (48 h) inoculation with the China isolate of white spot syndrome virus (WSSV). A total of 224 clones were sequenced, 194 of which were useful for homology comparisons against annotated genes in NCBI nonredundant (nr) and protein databases, providing 179 sequences encoded by nuclear DNA, 4 mitochondrial DNA, and 11 were similar to portions of WSSV genome. The nuclear sequences clustered in 43 groups, 11 of which were homologous to various ESTs of unknown function, 4 had no homology to any sequence, and 28 showed similarities to known genes of invertebrates and vertebrates, representatives of cellular metabolic processes such as calcium ion balance, cytoskeleton mRNAs, and protein synthesis. A few sequences were homologous to immune system-related (allergens) genes and two were similar to motifs of the sex-lethal gene of Drosophila. A large number of EST sequences were similar to domains of the EF-hand superfamily (Ca2+ binding motif and FRQ protein domain of myosin light chains). Single or multiple SSRs with three or more repeats were found in approximately 61 % of the 179 nuclear sequences. Primer sets were designed from 28 sequences representing 19 known or putative genes and tested for polymorphism (EST-SSR marker) in a small test panel containing 16 individuals. Ten (53%) of the 19 putative or unknown function genes were polymorphic, 4 monomorphic, and 3 either failed to satisfactorily amplify genomic DNA or the allele amplification conditions need to be further optimized. Five polymorphic ESTs were genotyped with the entire reference mapping family, two of them (actin, accession #CX535973 and shrimp allergen arginine kinase, accession #CX535999) did not amplify with all offspring of the IRMF panel suggesting presence of null alleles, and three of them amplified in most of the IRM F offspring and were used for linkage analysis. EF-hand motif of myosin light chain (accession #CX535935) was placed in ShrimpMap's linkage group 7, whereas ribosomal protein S5 (accession #CX535957) and troponin I (accession #CX535976) remained unassigned. Results indicate that (a) a large number of ESTs isolated from this cDNA library are similar to cytoskeleton mRNAs and may reflect a normal pathway of the cellular response after im infection with WSSV, and (b) primers flanking single or multiple SSRs with three or more repeats from shrimp ESTs could be an efficient approach to develop polymorphic markers useful for linkage mapping. Work is underway to map additional SSR-containing ESTs from this and other cDNA libraries as a plausible strategy to increase marker density in ShrimpMap.

Geophysical signatures associated with fluid flow and gas hydrate occurrence in a tectonically quiescent sequence, Qiongdongnan Basin, South China Sea

Interpretation of high-resolution two-dimensional (2D) and three-dimensional (3D) seismic data collected in the Qiongdongnan Basin, South China Sea reveals the presence of polygonal faults, pockmarks, gas chimneys and slope failure in strata of Pliocene and younger age. The gas chimneys are characterized by low-amplitude reflections, acoustic turbidity and low P-wave velocity indicating fluid expulsion pathways. Coherence time slices show that the polygonal faults are restricted to sediments with moderate-amplitude, continuous reflections. Gas hydrates are identified in seismic data by the presence of bottom simulating reflectors (BSRs), which have high amplitude, reverse polarity and are subparallel to seafloor. Mud diapirism and mounded structures have variable geometry and a great diversity regarding the origin of the fluid and the parent beds. The gas chimneys, mud diapirism, polygonal faults and a seismic facies-change facilitate the upward migration of thermogenic fluids from underlying sediments. Fluids can be temporarily trapped below the gas hydrate stability zone, but fluid advection may cause gas hydrate dissociation and affect the thickness of gas hydrate zone. The fluid accumulation leads to the generation of excess pore fluids that release along faults, forming pockmarks and mud volcanoes on the seafloor. These features are indicators of fluid flow in a tectonically-quiescent sequence, Qiongdongnan Basin. Geofluids (2010) 10, 351-368.

A close phylogenetic relationship between Sipuncula and Annelida evidenced from the complete mitochondrial genome sequence of Phascolosoma esculenta

Background: There are many advantages to the application of complete mitochondrial (mt) genomes in the accurate reconstruction of phylogenetic relationships in Metazoa. Although over one thousand metazoan genomes have been sequenced, the taxonomic sampling is highly biased, left with many phyla without a single representative of complete mitochondrial genome. Sipuncula (peanut worms or star worms) is a small taxon of worm-like marine organisms with an uncertain phylogenetic position. In this report, we present the mitochondrial genome sequence of Phascolosoma esculenta, the first complete mitochondrial genome of the phylum. Results: The mitochondrial genome of P. esculenta is 15,494 bp in length. The coding strand consists of 32.1% A, 21.5% C, 13.0% G, and 33.4% T bases (AT = 65.5%; AT skew = -0.019; GC skew = -0.248). It contains thirteen protein-coding genes (PCGs) with 3,709 codons in total, twenty-two transfer RNA genes, two ribosomal RNA genes and a non-coding AT-rich region (AT = 74.2%). All of the 37 identified genes are transcribed from the same DNA strand. Compared with the typical set of metazoan mt genomes, sipunculid lacks trnR but has an additional trnM. Maximum Likelihood and Bayesian analyses of the protein sequences show that Myzostomida, Sipuncula and Annelida (including echiurans and pogonophorans) form a monophyletic group, which supports a closer relationship between Sipuncula and Annelida than with Mollusca, Brachiopoda, and some other lophotrochozoan groups. Conclusion: This is the first report of a complete mitochondrial genome as a representative within the phylum Sipuncula. It shares many more similar features with the four known annelid and one echiuran mtDNAs. Firstly, sipunculans and annelids share quite similar gene order in the mitochondrial genome, with all 37 genes located on the same strand; secondly, phylogenetic analyses based on the concatenated protein sequences also strongly support the sipunculan + annelid clade (including echiurans and pogonophorans). Hence annelid "key-characters" including segmentation may be more labile than previously assumed.

Mitochondrial cytochrome b sequence variation and phylogenetics of the highly specialized schizothoracine fishes (Teleostei : Cyprinidae) in the Qinghai-Tibet Plateau

The complete 1140 bp mitochondial cytochrome b sequences were obtained from 39 individuals representing five species of all four genera of highly specialized schizothoracine fishes distributed in the Qinghai-Tibet plateau. Sequence variation of the cytochrome b gene was surveyed among the 39 individuals as well as three primitive schizothoracines and one outgroup. Phylogenetic analysis suggested that the group assignment based on 1140 bp of the cytochrome b sequence is obviously; different from previous assignments, and the highly specialized schizothoracine fishes (Schizopygopsis pylzovi, Gymnocypris przewalskii, G. eckloni, Chuanchia lablosa, and Platypharodon extremus) form a monophyletic group that is sister to the clade formed by the primitive schizothoracine fishes (Schizothorax prenanti, S. pseudaksaiensis, and S. argentatus). The haplotypes of Schizopygopsis pylzovi and G. przewalskii were paraphyletic based on cytochrome b data, which most likely reflected incomplete sorting of mitochondrial DNA lineages. The diploid chromosome numbers of Schizofhoracinae were considered in phylogenetic analysis and provided a clear pattern of relationships. Molecular dating estimated for highly specialized schizothoracine fishes suggested that the highly specialized schizothoracine fishes diverged in the late Miocene Pliocene to Pleistocene (4.5x10(4)-4.05x10(6) Years BP). The relationship between the cladogenesis of highly specialized schizothoracine fishes and geographical events of the Qinghai-Tibet plateau is discussed.

Phylogeography of the Qinghai-Tibetan Plateau endemic Juniperus przewalskii (Cupressaceae) inferred from chloroplast DNA sequence variation

The vegetation of the northeast Qinghai-Tibetan Plateau is dominated by alpine meadow and desert-steppe with sparse forests scattered within it. To obtain a better understanding of the phylogeography of one constituent species of the forests in this region, we examined chloroplast trnT-trnF and trnS-trnG sequence variation within Juniperus przewalskii, a key endemic tree species. Sequence data were obtained from 392 trees in 20 populations covering the entire distribution range of the species. Six cpDNA haplotypes were identified. Significant population subdivision was detected (G(ST) = 0.772, N-ST = 0.834), suggesting low levels of recurrent gene flow among populations and significant phylogeographic structure (N-ST > G(ST), P < 0.05). Eight of the nine disjunct populations surveyed on the high-elevation northeast plateau were fixed for a single haplotype (A), while the remaining, more westerly population, contained the same haplotype at high frequency together with two low frequency haplotypes (C and F). In contrast, most populations that occurred at lower altitudes at the plateau edge were fixed or nearly fixed for one of two haplotypes, A or E. However, two plateau edge populations had haplotype compositions different from the rest. In one, four haplotypes (A, B, D and E) were present at approximately equivalent frequencies, which might reflect a larger refugium in the area of this population during the last glacial period. Phylogenetic analysis indicated that the most widely distributed haplotype A is not ancestral to other haplotypes. The contrasting phylogeographic structures of the haplotype-rich plateau edge area and the almost haplotype-uniform plateau platform region indicate that the plateau platform was recolonized by J. przewalskii during the most recent postglacial period. This is supported by the findings of a nested clade analysis, which inferred that postglacial range expansion from the plateau edge followed by recent fragmentation is largely responsible for the present-day spatial distribution of cpDNA haplotypes within the species.

A new species of Saussurea (Asteraceae) from Tibet and its systematic position based on ITS sequence analysis

A new species of Saussurea, S. erecta S. W Liu, J. T Pan A J. Q. Liu sp. nov., is described from Tibet. It resembles S. kingii but may be distinguished by having distinct stems and glabrous achenes. Saussurea kingii was placed in sect. Pseudoeriocoryne of subgen. Eriocoryne; this section was circumscribed by acaulescence and an inflorescence with congested capitula surrounded by a rosette of leaves. The discovery of S. erecta with distinct stems, cauline leaves and corymbose capitula blurred the delimitation of sect. Pseudoeriocoryne and suggested that the section may be polyphyletic. Both the close relationship and the significant difference between S. erecta and S. kingii were confirmed by analyses of nrDNA ITS sequences. The resulting phylogenies based on ITS data further suggest that Saussurea sect. Pseudoeriocoryne, as traditionally defined, does not constitute a monophyletic group. The rapid radiation and speciation of Saussurea in the Qinghai-Tibetan Plateau, as inferred from ITS phylogeny, are discussed. (c) 2005 The Linnean Society of London.

Comonomer sequence distribution in metallocene-based ethylene/1-butene (S-34) and ethylene/1-hexene (S-43) copolymers

Metallocene based polyethylenes were prepared by SMOPEC's "metallocene adduct" technology in a gas phase fluidized bed model reactor. The C-13-NMR spectra of ethylene/1-butene (S-34) and ethylene/1-hexene(S-43) copolymers were studied in a manner analogous to that established by Hsieh and Cheng. The comonomer sequence distributions of copolymer samples were obtained. The results show that these metallocene based copolymers contain a small amount of butene and hexene, and the EE and EEE sequences are dominant.

Sequence-Seeking and Counter Streams: A Model for Information Processing in the Cortex

This paper presents a model for the general flow in the neocortex. The basic process, called "sequence-seeking," is a search for a sequence of mappings or transformations, linking source and target representations. The search is bi-directional, "bottom-up" as well as "top-down," and it explores in parallel a large numbe rof alternative sequences. This operation is implemented in a structure termed "counter streams," in which multiple sequences are explored along two separate, complementary pathways which seeking to meet. The first part of the paper discusses the general sequence-seeking scheme and a number of related processes, such as the learning of successful sequences, context effects, and the use of "express lines" and partial matches. The second part discusses biological implications of the model in terms of connections within and between cortical areas. The model is compared with existing data, and a number of new predictions are proposed.